Cytotoxicity of Local Anesthetics on Human Mesenchymal ......so, local anesthetics could both directly injure tissues within a joint and decrease the ability of tissues to heal. In

Cytotoxicity of Local Anesthetics on HumanMesenchymal Stem Cells

Ruyan Rahnama, BA, Miqi Wang, BA, Alexis C. Dang, MD, Hubert T. Kim, MD, PhD, and Alfred C. Kuo, MD, PhD

Investigation performed at the Department of Orthopaedic Surgery, University of California San Francisco, San Francisco,and the San Francisco Veterans Affairs Medical Center, San Francisco, California

Background: Local anesthetics are frequently delivered intra-articularly to provide perioperative pain control. Previousstudies have shown that the commonly used drugs lidocaine, ropivacaine, and bupivacaine can be toxic to humanchondrocytes. The present study was conducted to determine whether the toxic effects of local anesthetics on humanchondrocytes also extend to human mesenchymal stem cells.

Methods: Human mesenchymal stem cells from three healthy donors were grown in tissue culture and exposed to thefollowing anesthetic treatments for sixty minutes: (1) 1% lidocaine, (2) 2% lidocaine, (3) 0.25% bupivacaine, (4) 0.5% bupiv-acaine, (5) 0.2% ropivacaine, and (6) 0.5% ropivacaine. The cells were then allowed to recover for twenty-four hours in regulargrowth media, and viability was measured with use of fluorescent staining for live cells or a luminescence assay for ATP content.

Results: The live cell counts and ATP content were correlated (r2 = 0.79), and 2% lidocaine was found to be significantlymore toxic than all doses of bupivacaine and ropivacaine. Treatment with 1% lidocaine resulted in significantly fewer livecells (49%) compared with the control, and the live cell count was also significantly less than that for the other anesthetics.However, the ATP level in the 1% lidocaine group was not significantly lower than those in the other groups. Bupivacaineand ropivacaine did not exhibit significant differences in toxicity compared with the control or with each other.

Conclusions: Ropivacaine and bupivacaine had limited toxicity in human mesenchymal stem cells. However, lidocainecould significantly decrease mesenchymal stem cell viability. Since other studies have shown ropivacaine to be less toxicto chondrocytes than bupivacaine, ropivacaine may be a safer intra-articular anesthetic.

Clinical Relevance: Mesenchymal stem cells likely play a key role in healing following surgical procedures such asmicrofracture and ligament reconstruction. If local anesthetics are used following joint surgery, selection of an agent withlow toxicity toward mesenchymal stem cells, such as ropivacaine, may maximize tissue healing potential.

Local anesthetics are frequently delivered into joints toprovide postoperative analgesia. The intra-articular deliveryof local anesthetics after arthroscopy through single injec-

tions or through continuously acting pain pumps and indwellingcatheters has been shown to substantially minimize postoperativepain and maintain joint motion1-4. Such use of local anestheticsis a common practice worldwide. In one survey of all orthopaedicunits associated with the Swedish Arthroscopic Society, all butone of thirty-seven practices used intra-articular local anesthesiafor postoperative pain relief following knee arthroscopy5.

Despite their efficacy in pain control, local anestheticsmay also have detrimental effects. Numerous studies have

linked their use to the development of chondrolysis, a com-plication characterized by the rapid destruction of articularcartilage6-8. Notably, shoulder arthroscopy patients diagnosedwith rapid onset of glenohumeral chondrolysis routinely have ahistory of intra-articular infusion of bupivacaine or lidocaine8.Consistent with these clinical findings, numerous in vivo andin vitro studies have shown that common local anestheticssuch as bupivacaine, ropivacaine, and lidocaine can be toxic tochondrocytes. Such toxicity depends on the specific anesthetic,duration of exposure, and dose administered9-13. For example,0.5% ropivacaine induced less cell death after a thirty-minuteexposure in vitro compared with 0.5% bupivacaine in both

Disclosure: None of the authors received payments or services, either directly or indirectly (i.e., via his or her institution), from a third party in support of anyaspect of this work. None of the authors, or their institution(s), have had any financial relationship, in the thirty-six months prior to submission of this work,with any entity in the biomedical arena that could be perceived to influence or have the potential to influence what is written in this work. Also, no author hashad any other relationships, or has engaged in any other activities, that could be perceived to influence or have the potential to influence what is written inthis work. The complete Disclosures of Potential Conflicts of Interest submitted by authors are always provided with the online version of the article.

132

COPYRIGHT � 2013 BY THE JOURNAL OF BONE AND JOINT SURGERY, INCORPORATED

J Bone Joint Surg Am. 2013;95:132-7 d http://dx.doi.org/10.2106/JBJS.K.01291

isolated human articular chondrocytes and intact articularcartilage13.

Although the chondrotoxicity of local anesthetics hasbeen well studied, the effects of these drugs on reparative cellswithin a joint are unknown. Many orthopaedic procedures,including tendon repair, ligament reconstruction, and surgicalmarrow stimulation procedures such as microfracture, dependon patient healing responses. Mesenchymal stem cells likelyplay a key role in these processes. Under appropriate condi-tions, these cells can form bone, fat, tendon, and cartilage14,15.These regenerative cells are found in multiple tissues, includingbone marrow, adipose, and synovium14. In addition to thehealing resulting from the action of endogenous mesenchymalstem cells, surgical administration of exogenous mesenchymalstem cells may also enhance tissue regeneration16.

We hypothesized that the toxicity of local anesthetics onchondrocytes would also extend to mesenchymal stem cells. Ifso, local anesthetics could both directly injure tissues within ajoint and decrease the ability of tissues to heal. In this in vitrostudy, we evaluated the effects of commonly used doses oflidocaine, ropivacaine, and bupivacaine on the viability ofmesenchymal stem cells from three healthy donors. Our goalwas to identify treatments that minimized toxicity.

Materials and MethodsMesenchymal Stem Cell Monolayer Culture

Commercially available bone-marrow-derived human mesenchymal stemcells (Lonza, Walkersville, Maryland) from a twenty-year-old man (Lot

8F354), a thirty-year-old man (Lot 0F4452), and a twenty-two-year-old woman(Lot 0F3825) were plated in monolayer culture in high-glucose DMEM(Dulbecco’s modification of Eagle’s medium) (Mediatech, Manassas, Virginia)supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah) and 1%antibiotic/antimycotic solution (Hyclone) on tissue-culture-treated polystyreneat a density of 5000 cells/cm2.

The cells were grown at 37�C in a humidified 5% CO2 incubator until90% confluent, were trypsinized, and were expanded until the fifth or sixthpassage. They were then seeded onto ninety-six-well plates at a density of 2500cells/well (8000 cells/cm2) and cultured for forty-eight hours, leading to 90%confluence prior to experimental treatment.

Treatment GroupsMesenchymal stem cell cultures were subdivided into six replicate wells percondition and exposed to one of the following drug dilutions: (1) 2% lidocaine,pH 6 (APP Pharmaceuticals, Schaumburg, Illinois), (2) 1% lidocaine, pH 6, (3)0.5% bupivacaine, pH 6 (Hospira, Lake Forest, Illinois), (4) 0.25% bupivacaine,pH 6, (5) 0.5% ropivacaine, pH 5 (APP Pharmaceuticals), and (6) 0.2%ropivacaine, pH 5. The higher concentration of each anesthetic was used asprovided by the manufacturer. The supplied solutions were preservative-free,with sodium chloride (to adjust osmolarity) and sodium hydroxide or hydro-chloric acid (to adjust pH) added by the manufacturer. Control groups weretreated with 0.9% saline solution (APP Pharmaceuticals), which was also usedto prepare the lower concentration of lidocaine and of bupivacaine from thehigher concentration. The 0.2% ropivacaine solution was used as provided bythe manufacturer or diluted from 0.5% ropivacaine with 0.9% saline solution.

The cell culture media was aspirated, and 200 mL of the appropriatetreatment solution was added to each well. Cells were incubated in the treat-ment solution for sixty minutes at 37�C in a humidified 5% CO2 incubator,washed in 1· phosphate-buffered saline (PBS) solution, and returned to theincubator in fresh culture media. Mesenchymal stem cell viability was measuredtwenty-four hours later.

Assessment of ViabilityLive Cell CountsThe cell culture media was gently aspirated from each well, and viable mesen-chymal stem cells were stained with a LIVE/DEAD Viability/Cytotoxicity Kit (LifeTechnologies, Grand Island, New York). Briefly, 100 mL of a 1:2000 dilution ofcalcein AM (acetomethoxy) in PBS was added to each well. Cells were incubatedfor forty-five minutes at room temperature and then visualized with use of anAxiovert 200M fluorescent microscope (Carl Zeiss Microscopy, Thornwood, NewYork) with a fluorescein filter. Digital photographs of the center of each well weretaken at 5· magnification. The cell viability in each field was quantified by su-perimposing a grid onto the digital image in Photoshop (Adobe Systems, San Jose,California), and calcein-stained live cells, which exhibit green fluorescence, werecounted. The number of dead cells was also quantified in a subset of the ex-periments by staining with ethidium homodimer-1 and visualization with useof a rhodamine filter. Ethidium-stained dead cells and calcein-stained live cellswere counted, and the percentage of live cells for each treatment was calculated.

ATP (Adenosine Triphosphate) ContentThe ATP content in treated samples was measured with use of the CellTiter-GloLuminescent Cell Viability Assay (Promega, Madison, Wisconsin) accordingto the manufacturer’s instructions. Briefly, the CellTiter-Glo buffer and theCellTiter-Glo substrate were combined to form the CellTiter-Glo reagent. Thereagent was added to the culture media in each well in a 1:1 ratio, mixed well,and incubated for ten minutes at room temperature. Luminescence was thenmeasured with use of a Wallac 1420 VICTOR

2Multilabel Counter (PerkinElmer,

Waltham, Massachusetts).

Statistical AnalysisLive cell counts from the three independent trials, calculated as a percentage of thecontrol, were analyzed together with use of mixed-model analysis of variance(ANOVA), with the specified drug concentration as the fixed factor and trial as arandom factor. Live cell count results are reported for each drug concentration asthe least-squares mean (the calculated best-fit value across all trials) and thestandard error. ATP content results are reported for each of the three individualdonors as the mean luminescence (the CellTiter-Glo output) and the standarderror. Differences in ATP content among treatment groups were assessed for eachdonor with use of ANOVA. Results for the three donors were combined with useof mixed-model ANOVA, with the specified drug concentration as the fixed factorand donor as a random factor. ATP content results are reported as the least-squares mean luminescence and the standard error. Because multiple compari-sons were made, the significance of differences among treatments was calculatedwith use of the Tukey honestly significant difference test; a p value of <0.05 wasconsidered significant. Statistical analysis was performed with use of JMP(version 9.0; SAS Institute, Cary, North Carolina).

Source of FundingThis work was performed with funding from the Department of OrthopaedicSurgery of the University of California San Francisco and with resources of theVeterans Affairs Medical Center, San Francisco, California. There was no externalfunding source.

ResultsComparison of Cell Viability Measured with the Live CellCount, Percentage of Live Cells, and ATP Content

In a pilot experiment, mesenchymal stem cells from one of thedonors (the twenty-year-old male) were exposed to each of

the six anesthetic conditions, and viability was determined witheach of three methods: (1) live cell count, (2) percentage of livecells, and (3) ATP content. Six replicates were performed foreach of the three evaluation methods for each condition. Linearregression yielded an r2 value of 0.84 between the live cell countand the percentage of live cells and an r2 value of 0.79 between

133

TH E J O U R N A L O F B O N E & JO I N T SU R G E RY d J B J S . O R G

VO LU M E 95-A d NU M B E R 2 d JA N UA RY 16, 2013CY T O T OX I C I T Y O F LO C A L AN E S T H E T I C S O N HU M A N

ME S E N C H Y M A L ST E M CE L L S

the live cell count and the ATP content. Given the strength ofthese correlations, we evaluated viability with use of the livecell count and the ATP content for this donor and with useof only the ATP content for the other two donors.

Live Cell CountsThe number of live cells remaining after treatment with localanesthetic was determined with use of mesenchymal stem cellsfrom the twenty-year-old male donor. Treatment with 0.25% and0.5% bupivacaine in three independent trials reduced the live cell

count to 94% and 79%, respectively, of the value in the controlgroup. Treatment with 0.2% and 0.5% ropivacaine reduced thelive cell count to 83% and 84%, respectively, of the value in thecontrol group. No difference among these four groups wassignificant. Treatment with both concentrations of lidocaine,however, resulted in a significant reduction in the live cellcount compared with all other groups. Treatment with 1%lidocaine reduced the live cell count to 49% of the value inthe control group (p < 0.0001), and 2% lidocaine reduced itto 32% (p < 0.0001) (Figs. 1 and 2).

Fig. 1

Thenumberof livehumanmesenchymalstem

cells present twenty-four hours after a sixty-

minute treatment with 0.9% normal saline

solution (Control), 1% and 2% lidocaine (Lido),

0.25% and 0.5% bupivacaine (Bup), and 0.2%

and 0.5% ropivacaine (Rop). Three separate

experiments were performed in each treat-

ment group. The bar heights represent the

least-squares mean of the number of live cells

relative to the control, and the error bars rep-

resent the standard error of the mean. The

results for treatments labeled with different

letters (e.g., AandB)differedsignificantly from

each other (p < 0.05), whereas treatments

labeled with the same letter did not.

Fig. 2

Fluorescent microscopy images of human mes-

enchymal stem cells stained with calcein AM

(acetomethoxy) twenty-four hours after a sixty-

minute treatment with 0.9% normal saline solu-

tion (Control) (Fig. 2-A), 0.5% bupivacaine (Fig.

2-B), 0.5% ropivacaine (Fig. 2-C), and 1% lido-

caine (Fig. 2-D) (·5).

134




ATP ContentAs an alternative, independent measure of cell viability,cellular ATP content after local anesthetic treatment wasdetermined with use of mesenchymal stem cells from eachof the three donors (the twenty-year-old man, the twenty-two-year-old woman, and the thirty-year-old man). Theresults for each donor as well as the results of a modelcombining information from all three donors are shownin Figure 3. In all four cases, 2% lidocaine treatment sig-nificantly reduced viability compared with treatment witheither ropivacaine or bupivacaine. In the combined model,the ATP content in the 2% lidocaine group was only 49%of that in the 0.2% ropivacaine group. For the twenty-year-old donor, the ATP content in the 1% and 2% lido-caine groups was only 56% and 39%, respectively, of thatin the 0.2% ropivacaine group (p < 0.05). However, thedifference between the 1% lidocaine group and the othertreatment groups was not significant for the other twodonors or for the combined model. No significant differ-ences were found between the ropivacaine and bupivacainegroups.

Discussion

The chondrotoxicity of local anesthetics including bupiva-caine, ropivacaine, and lidocaine in both human and

animal models has been widely reported. However, to ourknowledge, the effects of these drugs on human mesenchymalstem cells have not been previously addressed. Given the likelyrole of these cells in tissue healing following orthopaedic pro-cedures, the toxicity of local anesthetics on these cells may beclinically important14. The present study indicated that bupiva-caine and ropivacaine at the commonly used 0.5% concentrationwere significantly less toxic than a concentration of lidocainewith equivalent anesthetic potency. These in vitro results sug-gest that intra-articular administration of lidocaine may havedetrimental effects on mesenchymal stem cell populations.

The relative toxicities of bupivacaine, ropivacaine, andlidocaine on mesenchymal stem cells differed from their tox-icities on human chondrocytes. Previous in vitro and in vivostudies have shown that bupivacaine is more toxic to cartilagecells than ropivacaine and lidocaine11,13,17. Specifically, Piper andKim directly compared the toxicity of 0.5% ropivacaine withthat of 0.5% bupivacaine and found that although ropivacaine

Fig. 3

Human mesenchymal stem cell viability twenty-four hours after a sixty-minute treatment with 1% and 2% lidocaine (Lido), 0.25% and0.5% bupivacaine (Bup),

and 0.2% and 0.5% ropivacaine (Rop). Viability was assessed on the basis of luminescence due to adenosine triphosphate (ATP), which was measured with

use of a CellTiter-Glo assay. The bar heights represent the least-squares mean for each treatment, and the error bars represent the standard error of the

mean. Results are presented for three individual donors (Figs. 3-A, 3-B, and 3-C) and for a model combining the three donors (Fig. 3-D). The results for

treatments labeledwith different letters (e.g., A and B) differed significantly from each other (p< 0.05), whereas treatments labeledwith the same letter did not.

135




did induce cell death in human chondrocytes, it was signifi-cantly less chondrotoxic than bupivacaine13. The observed cy-totoxicity differences between chondrocyte and mesenchymalstem cell populations suggest a differential effect of local an-esthetics on different cell types.

The inherent differences in physical and chemical pro-perties among these drugs likely contribute to the differences intheir relative toxicities on different cell types. Bupivacaine andropivacaine are highly lipophilic molecules, whereas lidocaineis only slightly lipophilic18,19. Treatment of human leukemiacells with these anesthetics induces different patterns of celldeath. Bupivacaine and ropivacaine predominantly cause ne-crosis, whereas lidocaine induces DNA fragmentation andapoptosis (programmed cell death)19. Studies of human T-celllymphoma and neuroblastoma cell lines showed that lidocainetreatment leads to apoptosis but ropivacaine does not. Thesedifferences may be due to differential activation of caspases,which are proteolytic enzymes that play key roles in apoptosis20,21.The three anesthetics may also differentially affect inflammation.Bupivacaine induces nitric oxide synthase-2 activity in rat glialcells and astrocytes but ropivacaine does not22. This pathway isnormally activated as part of an inflammatory response, sug-gesting that bupivacaine toxicity may be attributable in part tothe production of nitric oxide during ongoing inflammation.In contrast, ropivacaine has anti-inflammatory properties13.

The relative potencies of lidocaine, bupivacaine, andropivacaine were accounted for in our comparisons among thedrugs. Bupivacaine and ropivacaine are highly potent anestheticswith long-lasting effects, whereas lidocaine is a moderately po-tent anesthetic with shorter effects23. Specifically, bupivacaineand ropivacaine are approximately four times more potent an-esthetics than lidocaine24. Therefore, the effects of 0.5% bupiva-caine and 0.5% ropivacaine on pain should be similar to that of2% lidocaine, but with a longer duration of action and less toxicityon mesenchymal stem cells. Although the CellTiter-Glo assay forATP content was slightly less sensitive to differences amongconditions than the live cell count, similar trends were found withboth methods of analysis.

One limitation of the present study is the fact that theexperiments were performed in vitro. Further experiments areneeded to assess the in vivo effects of these drugs on mesenchymalstem cell viability. However, the experimental conditions in thepresent study (growth of cells in tissue culture to yield a mono-layer in the fifth or sixth passage) are commonly used in studies ofmesenchymal stem cells25,26, and such cells are defined in part by

their ability to adhere, proliferate, and differentiate into mesen-chymal tissues under such in vitro conditions27. Another limita-tion is the fact that viability measurements were only performedafter sixty minutes of anesthetic treatment. This duration of ex-posure was chosen on the basis of a previous study of the phar-macokinetics of bupivacaine after intra-articular injection into theknee28. The absorption of this anesthetic from the joint into thebloodstream is rapid, with the peak blood concentration beingreached within the first hour after injection. We are not aware ofsimilar data for lidocaine or ropivacaine. We did not test for timedependence or for potential early-acting toxicity in the drugtreatments. The effect of local anesthetic treatment on the dif-ferentiation potential of mesenchymal stem cells is also of interestin future experiments. Although cells treated with bupivacaineand ropivacaine in the present study appeared to be metabolicallyand morphologically normal, the drugs may subtly affect dif-ferentiation patterns. To study these effects, mesenchymal stemcells treated with varying classes and doses of local anesthetic canbe assayed for the ability to form bone, cartilage, and fat.

In conclusion, in contrast to the toxicity observed in hu-man and bovine chondrocytes, commonly used concentrationsof ropivacaine and bupivacaine appeared to have limited toxicityon human mesenchymal stem cells. In contrast, lidocaine sig-nificantly decreased mesenchymal stem cell viability. Althoughthese experiments are limited by their in vitro nature, these datasuggest that ropivacaine and bupivacaine may represent betteroptions with respect to mesenchymal stem cell health. Takinginto further account the chondrotoxicity of bupivacaine andthe chondrolysis associated with postoperative bupivacaineinfusions, ropivacaine may be the safest of these three choicesfor intra-articular analgesia. n

NOTE: The authors thank Dr. Eva Grotkopp for statistical assistance and Linsey Jacobs and Dr.Sonja Limburg for technical assistance.

Ruyan Rahnama, BAMiqi Wang, BAAlexis C. Dang, MDHubert T. Kim, MD, PhDAlfred C. Kuo, MD, PhDDepartment of Orthopaedic Surgery,University of California San Francisco,San Francisco Veterans Affairs Medical Center,4150 Clement Street Box 112,San Francisco, CA 94121

References

1. Chirwa SS, MacLeod BA, Day B. Intraarticular bupivacaine (Marcaine) after ar-throscopic meniscectomy: a randomized double-blind controlled study. Arthroscopy.1989;5(1):33-5.2. Katz JA, Kaeding CS, Hill JR, Henthorn TK. The pharmacokinetics of bupivacainewhen injected intra-articularly after knee arthroscopy. Anesth Analg. 1988Sep;67(9):872-5.3. Middleton F, Coakes J, Umarji S, Palmer S, Venn R, Panayiotou S. The efficacy ofintra-articular bupivacaine for relief of pain following arthroscopy of the ankle. J BoneJoint Surg Br. 2006 Dec;88(12):1603-5.4. Yamaguchi K, Sethi N, Bauer GS. Postoperative pain control following arthro-scopic release of adhesive capsulitis: a short-term retrospective review study of theuse of an intra-articular pain catheter. Arthroscopy. 2002 Apr;18(4):359-65.

5. Brattwall M, Jacobson E, Forssblad M, Jakobsson J. Knee arthroscopy routinesand practice. Knee Surg Sports Traumatol Arthrosc. 2010 Dec;18(12):1656-60.Epub 2010 Sep 21.6. Anderson SL, Buchko JZ, Taillon MR, Ernst MA. Chondrolysis of the glenohumeraljoint after infusion of bupivacaine through an intra-articular pain pump catheter: areport of 18 cases. Arthroscopy. 2010 Apr;26(4):451-61.7. Busfield BT, Romero DM. Pain pump use after shoulder arthroscopy as a cause ofglenohumeral chondrolysis. Arthroscopy. 2009 Jun;25(6):647-52.8. Scheffel PT, Clinton J, Lynch JR, Warme WJ, Bertelsen AL, Matsen FA 3rd.Glenohumeral chondrolysis: a systematic review of 100 cases from the Englishlanguage literature. J Shoulder Elbow Surg. 2010 Sep;19(6):944-9. Epub 2010Apr 24.

136




9. Chu CR, Coyle CH, Chu CT, Szczodry M, Seshadri V, Karpie JC, Cieslak KM,Pringle EK. In vivo effects of single intra-articular injection of 0.5% bupivacaine onarticular cartilage. J Bone Joint Surg Am. 2010 Mar;92(3):599-608.10. Chu CR, Izzo NJ, Coyle CH, Papas NE, Logar A. The in vitro effects of bupivacaineon articular chondrocytes. J Bone Joint Surg Br. 2008 Jun;90(6):814-20.11. Karpie JC, Chu CR. Lidocaine exhibits dose- and time-dependent cytotoxic ef-fects on bovine articular chondrocytes in vitro. Am J Sports Med. 2007Oct;35(10):1621-7. Epub 2007 Jul 30.12. Lo IK, Sciore P, Chung M, Liang S, Boorman RB, Thornton GM, Rattner JB,Muldrew K. Local anesthetics induce chondrocyte death in bovine articular cartilagedisks in a dose- and duration-dependent manner. Arthroscopy. 2009 Jul;25(7):707-15.13. Piper SL, Kim HT. Comparison of ropivacaine and bupivacaine toxicity in humanarticular chondrocytes. J Bone Joint Surg Am. 2008 May;90(5):986-91.14. Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and bio-logical characterization. Int J Biochem Cell Biol. 2004 Apr;36(4):568-84.15. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD,Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adulthuman mesenchymal stem cells. Science. 1999 Apr 2;284(5411):143-7.16. Wilke MM, Nydam DV, Nixon AJ. Enhanced early chondrogenesis in articulardefects following arthroscopic mesenchymal stem cell implantation in an equinemodel. J Orthop Res. 2007 Jul;25(7):913-25.17. Piper SL, Kramer JD, Kim HT, Feeley BT. Effects of local anesthetics on articularcartilage. Am J Sports Med. 2011 Oct;39(10):2245-53. Epub 2011 Apr 22.18. Grouselle M, Tueux O, Dabadie P, Georgescaud D, Mazat JP. Effect of localanaesthetics on mitochondrial membrane potential in living cells. Biochem J. 1990Oct 1;271(1):269-72.19. Onizuka S, Yonaha T, Tsuneyoshi I. Local anesthetics with high lipophilicity aretoxic, while local anesthetics with low pka induce more apoptosis in human leukemiacells. J Anesth Clin Res. 2011;2:1.

20. Boselli E, Duflo F, Debon R, Allaouchiche B, Chassard D, Thomas L,Portoukalian J. The induction of apoptosis by local anesthetics: a comparison be-tween lidocaine and ropivacaine. Anesth Analg. 2003 Mar;96(3):755-6, table ofcontents.21. Perez-Castro R, Patel S, Garavito-Aguilar ZV, Rosenberg A, Recio-Pinto E,Zhang J, Blanck TJ, Xu F. Cytotoxicity of local anesthetics in human neuronal cells.Anesth Analg. 2009 Mar;108(3):997-1007.22. Feinstein DL, Murphy P, Sharp A, Galea E, Gavrilyuk V, Weinberg G. Localanesthetics potentiate nitric oxide synthase type 2 expression in rat glial cells.J Neurosurg Anesthesiol. 2001 Apr;13(2):99-105.23. Tomin J, Zivanov-Curlis J, Popovic D, Glogovac S, Basic D. Differences in localanesthetic effects of optically active isomers of local anesthetic compounds. Bio-technol Biotechnol Eq. 2006;20:9-14.24. Adams HR, editor. Veterinary pharmacology and therapeutics. 8th ed. Ames, IA:Iowa Stae University Press; 2001. p 346.25. Bian L, Zhai DY, Tous E, Rai R, Mauck RL, Burdick JA. Enhanced MSC chondro-genesis following delivery of TGF-b3 from alginate microspheres within hyaluronic acidhydrogels in vitro and in vivo. Biomaterials. 2011 Sep;32(27):6425-34. Epub 2011 Jun 8.26. Cooke ME, Allon AA, Cheng T, Kuo AC, Kim HT, Vail TP, Marcucio RS,Schneider RA, Lotz JC, Alliston T. Structured three-dimensional co-culture ofmesenchymal stem cells with chondrocytes promotes chondrogenic differentia-tion without hypertrophy. Osteoarthritis Cartilage. 2011 Oct;19(10):1210-8. Epub2011 Jul 23.27. Penfornis P, Pochampally R. Isolation and expansion of mesenchymal stemcells/multipotential stromal cells from human bone marrow. Methods Mol Biol.2011;698:11-21.28. Katz JA, Kaeding CS, Hill JR, Henthorn TK. The pharmacokinetics of bupivacainewhen injected intra-articularly after knee arthroscopy. Anesth Analg. 1988Sep;67(9):872-5.

137




Documents

Cytotoxicity of Local Anesthetics on Human Mesenchymal ......so, local anesthetics could both directly injure tissues within a joint and decrease the ability of tissues to heal. In